The present invention relates to methods for the production of polynucleotides conferring a desired phenotype and/or encoding a polypeptide having an advantageous predetermined property which is selectable or can be screened for.
Traditional molecular biological methods for generating novel genes and proteins generally involved rational or directed mutation. An example is the generation of a polynucleotide encoding a fusion or chimeric protein by using known restriction sites to combine functional domains from two characterized proteins. Another example is the introduction of a point mutation at a specific site in a polypeptide. Although useful, the power of these and similar methods is limited by the requirement for sequence or restriction map information to facilitate the mutagenesis, and by the limited number of variants that can be efficiently generated.
An alternative approach to the generation of variants uses random recombination techniques such as xe2x80x9cDNA shufflingxe2x80x9d (Patten et al., 1997, Curr. Opin. Biotech. 18:724-733). DNA shuffling entails performing iterative cycles of recombination and screening or selection to xe2x80x9cevolvexe2x80x9d individual genes, whole plasmids or viruses, multigene clusters, or whole genomes. Such techniques do not require the extensive analysis and computation required by conventional methods for engineering of polynucleotides and polypeptides. Moreover, DNA shuffling allows the recombination of large numbers of mutations in a minimum number of selection cycles, in contrast to traditional, pairwise recombination events. Thus, DNA shuffling techniques provide advantages in that they provide recombination between mutations in any or all of these, thereby providing a very fast way of exploring the manner in which different combinations of mutations can affect a desired result.
The present invention provides methods that may be used alone or in combination with random recombination techniques such as DNA shuffling to generate novel polynucleotides having, or encoding a polypeptide having, a desired property or combination of properties.
In one aspect, the invention provides a method of producing a DNA segment having a desired property or combination of properties by mutating a substrate population. The method involves:
a) mutating a substrate population that includes a plurality of DNA segments by:
i) making insertions at random sites in the segments (random insertion),
ii) making deletions at random sites in the segments (random deletion), or both, to produce a mutated population including mutated DNA segments,
b) screening the mutated population to obtain a first selected population that includes at least one DNA segment with a first desired property,
c) mutating the first selected population by making random insertions, random deletions, or both, to produce a recursively mutated population, and,
d) screening the recursively mutated population to obtain a recursively selected population that includes at least one DNA segment with a second desired property.
In some embodiments the method further includes at least one additional cycle of mutation and screening (e.g., mutating the recursively selected population and screening the resulting recursively mutated population to obtain new recursively selected population with a desired property) after step (d). In some embodiments, shuffling of one or a combination of polynucleotides in a recursively selected population is carried out.
In various embodiments, the second desired property may be the same or different from the first desired property, and may be a combination of properties. In some embodiments, the polynucleotides in the recursively selected population have a property that is enhanced when compared to the polynucleotides in the first selected population. In some embodiments the substrate population includes DNA segments encoding a polypeptide, a catalytic RNA, a promoter sequence or a vector. In some embodiments the substrate population is homogeneous. In some embodiments a polynucleotide that encodes a polypeptide is screened for an activity such as an enzymatic activity, a substrate specificity, or a binding activity of a polypeptide.
In another aspect, the invention provides a method of producing a DNA segment having a desired property by:
a) mutating a first substrate population that includes a plurality of DNA segments by:
i) making insertions at random sites in the segments (random insertion),
ii) making deletions at random sites in the segments (random deletion), or both, to produce a first mutated population of mutated DNA segments;
b) mutating a second substrate population that includes a plurality of DNA segments by:
i) making insertions at random sites in the segments,
ii) making deletions at random sites in the segments, or both to produce a second mutated population of mutated DNA segments;
c) recombining the first substrate population and the second substrate population to produce a recombined population; and,
d) screening the recombined population to identify at least one DNA segment with the desired property.
In one embodiment, the first and second mutated populations are screened to produce a first and second selected population, each having a desired property, and the selected populations are recombined.
In various embodiments, the recombination may be achieved by shuffling or directed recombination. In some embodiments the first desired property and the second desired property are the same. In some embodiments the substrate population includes DNA segments encoding a polypeptide, a catalytic RNA, a promoter sequence or a vector. In some embodiments the substrate population is homogeneous. In some embodiments a polynucleotide that encodes a polypeptide is screened for an activity such as an enzymatic activity, a substrate specificity, or a binding activity of a polypeptide.
In another aspect, the invention provides a method of producing a DNA segment having a desired property by:
a) mutating a substrate population that includes a plurality of DNA segments by:
i) making insertions at random sites in the segments,
ii) making deletions at random sites in the segments; or both, to produce a mutated population of mutated DNA segments;
b) screening the mutated population to obtain a selected population that includes at least one DNA segment with the desired property;
c) shuffling at least one DNA segment for the selected population to produce a recombined population;
d) screening the recombined population for a desired property.
In one embodiment, the shuffling involves conducting a polynucleotide amplification process on overlapping segments of at least one polynucleotide from the selected population under conditions under which one segment serves as a template for extension of another segment, to generate a population of recombinant polynucleotides.
In some embodiments the substrate population includes DNA segments encoding a polypeptide, a catalytic RNA, a promoter sequence or a vectors. In some embodiments the substrate population is homogeneous. In some embodiments a polynucleotide that encodes a polypeptide is screened for an activity such as an enzymatic activity, a substrate specificity, or a binding activity of a polypeptide.